Human growth hormone (hGH) is a human-specific hormone secreted by the anterior pituitary gland. The hormone is critical to the normal growth of children and is also significantly involved in regulating metabolism in both children and adults. Recombinant hGH is used in replacement therapy to treat conditions which produce short stature either due to hGH deficiency or due to other causes, such as Turner syndrome, Prader-Willi syndrome or intrauterine growth retardation.
Aqueous liquid compositions of hGH are very convenient and particularly suitable for convenient administration of the hormone by a pre-filled syringe or an injection pen. Several stabilized aqueous compositions of hGH have been disclosed based on the surprising observation that the presence of phenolic preservatives, particularly phenol, has a beneficial effect on stability of aqueous hGH. EP0955062 and WO2004/004780 disclose aqueous formulations of human growth hormone comprising a phenolic preservative. Whilst a range of phenolic preservatives, such as m-cresol, phenol, benzyl alcohol or benzethonium chloride, can be used according to EP0955062, WO2004/004780 is limited to phenol. The use of phenol as a preservative is claimed in WO2004/004780 to have been surprisingly provided improved stability of formulations of high concentrations of human growth hormone. Such formulations have been found to have a good resistance to crystallisation, i.e. formation of soluble and insoluble aggregates, over time. The most preferred concentration of phenol both in EP0955062 and in WO2004/004780 is 2.5 mg/mL, i.e. about 27 mM.
Apart from the use of phenolic compounds to improve stability of hGH, several other disclosures have been made describing stabilized liquid compositions of hGH. WO2005/027960 discloses compositions of hGH in the presence of polyethylene glycol (typically used at 10 mg/mL). Such formulations were demonstrated to reduce aggregation of hGH induced by agitation. At a concentration of hGH of 5 mg/mL, the protective effect was better than that observed in the presence of Poloxamer 188, a commonly used detergent in therapeutic formulations. The presence of polyethylene glycol was also shown to reduce to some extent chemical damage, such as deamidation during storage at 2-8° C. and 25° C. US2007/0014818 discloses hGH compositions in the presence of 1,2-propylene glycol showing reduced rate of crystallization as assessed by visual inspection compared with control compositions in the absence of 1,2-propylene glycol.
The key degradation pathways during storage of hGH, especially in aqueous liquid formulations, are deamidation and aggregation. The rate of deamidation is not significantly dependent on the concentration of the protein, and it can be minimized by keeping the pH of the formulation close to pH 6.1. The deamidation rate increases particularly at higher pH values wherein the alkaline deamidation process becomes prevalent. At lower pH values, it is a different mechanism of acidic deamidation which drives the conversion of amide groups of asparagine and glutamine to the respective carboxylic acids. The deamidated species of hGH are eqipotent with the native form of the protein and are not known to be more immunogenic than the native form. Nevertheless, the deamidation rate in the aqueous compositions of hGH should be kept at a minimum.
Whilst minimizing the rate of deamidation in therapeutic liquid compositions of hGH is of importance, it is critical to reduce the rate of aggregation processes. Formation of aggregates, both soluble and insoluble, increases significantly the risk of immunogenicity and strict limits are therefore imposed on the level of aggregates in such compositions. Aggregation is a process which is very dependent on the concentration of the protein. Consequently, aggregation is a particular problem in aqueous compositions comprising high concentration of hGH. Reducing the aggregation rate is therefore of particular importance in the case of high-concentration hGH compositions, such as compositions comprising >6 mg/mL and particularly >9 mg/mL of hGH.
The concentrations of hGH used in currently marketed liquid compositions are 3.3 mg/mL, 6.7 mg/mL or 10 mg/mL. It is very desirable to enable storage of such compositions at room temperature (<25° C.) at least for several days or ideally for several weeks. Whilst it is possible to ensure such stability at the lower end of the concentration range, the high rate of aggregation at the higher end of the concentration range (e.g., around 10 mg/mL) prevents the room temperature storage, even for a short period of time.
In order to receive regulatory approval for storage for a period of time at temperatures up to 25° C., it is essential to demonstrate acceptably low aggregation rate at 40° C. Accelerated stability trials at 40° C. are therefore a very important part of the stability assessment prior to approval. Typically, two parameters are used to assess acceptable aggregation rate in liquid formulations: (1) no particulate formation assessed either by eye or by a light scattering technique, and (2) <4% content of soluble high-molecular weight species (HMWS).
WO2007/003936 discloses stable compositions of proteins based on the presence of stabilizing agents characterized in that the one or more stabilizing agents have ionizable groups capable of exchanging protons with the protein, and the ionizable groups include first group that is positively charged when protonated and uncharged when deprotonated, and second group that is uncharged when protonated and negatively charged when deprotonated, the pKa of these ionizable groups being between 1 to 3 pH units from the pH of the composition.
WO2008/084237 describes how to improve, the stability of aqueous protein solutions. The disclosure is based on the discovery that buffers having a pKa at or near the pH of the solution are undesirable, when considering the protein's stability with respect to pH. Rather, the key to the invention is choice of the appropriate pH while minimizing the protein's ability to exchange hydrogen cations. In particular, an aqueous system is disclosed, which comprises a protein and one or more additives, characterized in that                (i) the system is substantially free of a conventional buffer, i.e a compound with a pKa within 1 unit of the pH of the composition at the intended temperature range of storage of the composition;        (ii) the pH of the composition is set to a value at which the composition has maximum measurable stability with respect to pH;        (iii) the one or more additives (displaced buffers) are capable of exchanging protons with the protein and have pKa values at least 1 unit more or less than the pH of the composition at the intended temperature range of storage of the composition.        
By keeping a protein at a suitable pH, at or near a value at which the measurable stability is maximal, in the absence of a conventional buffer, the storage stability of the protein can be increased substantially. Storage stability can generally be enhanced further, possibly substantially, by use of additives having pKa between 1 to 5 pH units, preferably between 1 to 3 pH units, most preferably between 1.5 to 2.5 pH units of the pH of the aqueous composition at the intended temperature range of storage of the composition. The presence of these additives also improves the pH stability of the formulation and is generally preferred.